The physiological modification of molecules and supramolecular assemblies plays a major role in the structure and regulation of biological systems. These modifications may include phosphorylation, cyclization, glycosylation, acylation, and/or sulfation, among others, and the modified molecules may include polypeptides, nucleic acids, and/or lipids, among others. The importance of modifications is particularly evident in the cell-signaling pathway, in which extracellular and intracellular substances related by phosphate modifications such as phosphorylation and cyclization influence the position, nature, and activity of cells.
Compounds which can interfere with phosphorylation or dephosphorylation catalysed by specific enzymes, or of specific substrates, are of interest since they may interfere with specific signaling events and thus be useful for treatment or prevention of diseases which occur through dysregulation of such pathways.
Therefore, it is of interest to develop methods of determining kinase or phosphatase activity for screening for compounds that can modulate specific signaling pathways.
EP1156329 discloses a method of determining kinase activity based on a system of two parts, the binding partner (BP) and the substrate molecule (A) which is converted (A*) or A->A*. In general, the binding partner is an entity that binds the enzyme product and can itself be attached to a solid phase e.g. a bead by any kind of interaction, covalent or non-covalent. For detection, a label (fluorophore, luminophore, acceptor or quencer) is attached to A or the BP. The list of detection methods includes intensity, polarization and energy transfer measurements.
WO00/75167 discloses a method of detecting addition or removal of a substrate group to or from a substrate which is also based on a system of two parts, the binding partner, which is preferably a macromolecule having entrapped metal ions, and a peptide A, wherein the conversion of phosphorylated to unphosphorylated peptide or vice versa is measured, e.g. by FRET (Fluorescence resonance energy transfer).
One drawback of the two parts systems is that for optimal energy transfer, for a given BP and A* ratio, a certain ratio of donor and acceptor is required. Thus, for optimal FRET generation, it may be necessary to titrate BP, A and A*.
However, the optimum ratios between BP, A and A* are generally influenced by the binding capacity of BP for A* and the respective binding constants on one hand, and also by the kinetic parameters of the enzyme reaction for the target of interest on the other hand. Therefore, the fixed ratio of BP, A and A* which is necessary for FRET optimization in a two parts system leads to a compromise in either assay signal and quality or enzyme kinetics and biological meaning of the results, i.e. in the worst case it may be impossible in a two parts system to achieve the optimum for all components: enzyme reaction for optimum kinetics, BP and A* for optimum binding of converted product, acceptor-donor for optimum FRET.
One embodiment of the two parts system disclosed in EP 1156329 and WO00/75167 comprises plastic beads that are doped with either luminophore or acceptor and a surface coating to bind the labeled A*. In other words IMAP™ beads with optical label in the core of the beads.
To achieve sufficient binding capacity the IMAP™ beads typically have a diameter of 100 nm or more. This is big compared to the energy transfer distance of a typical donor acceptor pair which is 3 to maximally 9 nm. As a consequence the energy transfer between a doped bead and a bound peptide is rather inefficient as there are many luminophores inside the bead which don't see a FRET partner but still contribute to assay background signal. This leads to a reduced dynamic range and decrease in sensitivity.
Therefore, there is a need to develop a more efficient method for determining kinase or phosphatase activity by energy transfer measurements between an energy donor and an energy acceptor